The Efg1-Bud22 dimer associates with the U14 snoRNP contacting the 5' rRNA domain of an early 90S pre-ribosomal particle.

The DEAD-box helicase Dbp4 plays an essential role during the early assembly of the 40S ribosome, which is only poorly understood to date. By applying the yeast two-hybrid method and biochemical approaches, we discovered that Dbp4 interacts with the Efg1-Bud22 dimer. Both factors associate with early pre-90S particles and smaller complexes, each characterized by a high presence of the U14 snoRNA. A crosslink analysis of Bud22 revealed its contact to the U14 snoRNA and the 5' domain of the nascent 18S rRNA, close to its U14 snoRNA hybridization site. Moreover, depletion of Bud22 or Efg1 specifically affects U14 snoRNA association with pre-ribosomal complexes. Accordingly, we concluded that the role of the Efg1-Bud22 dimer is linked to the U14 snoRNA function on early 90S ribosome intermediates chaperoning the 5' domain of the nascent 18S rRNA. The successful rRNA folding of the 5' domain and the release of Efg1, Bud22, Dpb4, U14 snoRNA and associated snoRNP factors allows the subsequent recruitment of the Kre33-Bfr2-Enp2-Lcp5 module towards the 90S pre-ribosome.

Molecular Apomorphies in the Secondary and Tertiary Structures of Length-Variable Regions (LVRs) of 18S rRNA Shed Light on the Systematic Position of the Family Thaumastellidae (Hemiptera: Heteroptera: Pentatomoidea).

The SSU nrDNA, a small subunit of the nuclear ribosomal DNA (coding 18S rRNA), is one of the most frequently sequenced genes in molecular studies in Hexapoda. In insects, including true bugs (Hemiptera: Heteroptera), only its primary structures (i.e., aligned sequences) are predominantly used in phylogenetic reconstructions. It is known that including RNA secondary structures in the alignment procedure is essential for improving accuracy and robustness in phylogenetic tree reconstruction. Moreover, local plasticity in rRNAs might impact their tertiary structures and corresponding functions. To determine the systematic position of Thaumastellidae within the superfamily Pentatomoidea, the secondary and-for the first time among all Hexapoda-tertiary structures of 18S rRNAs in twelve pentatomoid families were compared and analysed. Results indicate that the shapes of the secondary and tertiary structures of the length-variable regions (LVRs) in the 18S rRNA are phylogenetically highly informative. Based on these results, it is suggested that the Thaumastellidae is maintained as an independent family within the superfamily Pentatomoidea, rather than as a part of the family Cydnidae. Moreover, the analyses indicate a close relationship between Sehirinae and Parastrachiidae, expressed in morpho-molecular synapomorphies in the predicted secondary and tertiary structures of the length-variable region L (LVR L).

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.

Synergistic interaction network between the snR30 RNP, Utp23, and ribosomal RNA during ribosome synthesis.

snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the in vitro reconstitution of a Saccharomyces cerevisiae snR30 RNP and quantitative characterization of the interactions of snR30, H/ACA proteins, the Utp23 protein and ES6 of the 18S rRNA. The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.

Atypical U3 snoRNA Suppresses the Process of Pterygium Through Modulating 18S Ribosomal RNA Synthesis.

Background: The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development. Methods: Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in pterygium promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5' external transcribed spacer (5' ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusions: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.

Phylogeny of Lithobiidae Newport, 1844, with emphasis on the megadiverse genus Lithobius Leach, 1814 (Myriapoda, Chilopoda).

Phylogenetic analyses based on molecular and morphological data were conducted to shed light on relationships within the mostly Palaearctic/Oriental centipede family Lithobiidae, with a particular focus on the Palaearctic genus Lithobius Leach, 1814 (Lithobiidae, Lithobiomorpha), which contains >500 species and subspecies. Previous studies based on morphological data resolved Lithobius as nonmonophyletic, but molecular-based phylogenetic analyses have until now sampled few species. To elucidate species inter-relationships of the genus, test the validity of its classification into subgenera, and infer its relationships with other Lithobiidae, we obtained molecular data (nuclear markers: 18S rRNA, 28S rRNA; mitochondrial markers: 16S rRNA, COI) and 61 morphological characters for 44 species of Lithobius representing four of its eight subgenera and nine other representatives of Lithobiidae. The data were analyzed phylogenetically using maximum-likelihood, parsimony and Bayesian inference. This study suggests that (i) a close relationship between L. giganteus and the pterygotergine Disphaerobius loricatus highlighted in recent morphological analyses is also strongly supported by molecular data, and Pterygoterginae is formally synonymized with Lithobiinae; (ii) the Oriental/Australian genus Australobius is consistently resolved as sister group to all other sampled Lithobiidae by the molecular and combined data; (iii) the subfamily Ethopolyinae may be paraphyletic; (iv) the genus Lithobius is nonmonophyletic; (v) the subgenera Lithobius, Sigibius and Monotarsobius are nonmonophyletic and should not be used in future taxonomic studies; and (vi) there are instances of cryptic species and cases in which subspecies should be elevated to full species status, as identified for some European taxa within Lithobius.

A Case Report of Avian Malaria (Plasmodium spp.) in Pen-Reared Pigeons (Columba livia).

One dead 6-wk-old male racing pigeon (Columba livia) was submitted for postmortem evaluation after presenting with weight loss, anorexia, dry shanks, dehydration, and lethargy. The bird belonged to a confined flock with 12 other pigeons raised by a hobbyist. Two pigeons in the flock reportedly had died with a history of similar clinical signs. On gross examination, the liver and the spleen were diffusely dark brown to black. Histopathology revealed moderate to large amounts of anisotropic, intracytoplasmic black pigment, compatible with hemozoin, in the spleen, liver, lung, and kidneys, with small amounts in the heart and meninges of the brain. Marked plasmacytic infiltrates were observed in liver, lungs, heart, and kidneys. Blood smears from a clinically affected concomitant pigeon from the flock revealed numerous light-blue, round to oval, intraerythrocytic trophozoites and meronts suggestive of Plasmodium spp. PCR and sequencing tests were performed from spleen and ceca with fragments of the 18S ribosomal RNA and the mitochondrial cytochrome b (cytB) genes. Sequencing results confirmed the presence of Plasmodium in the affected pigeon. Although an exact genetic match could not be determined, the most similar species to the isolate from this study are Plasmodium relictum, Plasmodium matutinum, Plasmodium lutzi, and Plasmodium homocircumflexum.

Reporte de caso­Reporte de un caso de malaria aviar (Plasmodium spp.) en palomas criadas en corrales (Columba livia) Una paloma mensajera macho de 6 semanas muerta (Columba livia) fue remitido a evaluación post mortem después de presentar pérdida de peso, anorexia, patas secas, deshidrataciœn y letargo. El pájaro pertenecía a una parvada confinada con otras 12 palomas criadas por un criador aficionado. Dos palomas de la parvada habían muerto con antecedentes de signos clínicos similares. En el examen macroscópico, el hígado y el bazo se observaron de color marrón oscuro a negro. La histopatología reveló cantidades moderadas a abundantes de pigmento negro intracitoplasmático y anisotrópico, compatible con hemozoína, en el bazo, hígado, pulmón y riñones, con pequeñas cantidades en el corazón y en las meninges del cerebro. Se observaron marcados infiltrados plasmocíticos en hígado, pulmones, corazón y riñones. Los frotis de sangre de otra paloma clínicamente afectada de la parvada revelaron numerosos trofozoítos intraeritrocíticos y esquizontes de color azul claro, redondos a ovalados, que sugerían Plasmodium spp. Se realizaron pruebas de PCR y secuenciación a partir del bazo y el ciego con fragmentos de los genes de ARN ribosómico 18S y del citocromo b mitocondrial (cytB). Los resultados de la secuenciación confirmaron la presencia de Plasmodium en la paloma afectada. Aunque no se pudo determinar una identidad genética exacta, las especies más similares al aislado de este estudio son Plasmodium relictum, Plasmodium matutinum, Plasmodium lutzi y Plasmodium homocircumflexum.

First report of marine horsehair worms (Nematomorpha: Nectonema) parasitic in isopod crustaceans.

Nectonema, the only horsehair worm (Nematomorpha) genus found in marine environments, was previously known to be parasitic only in decapod crustaceans. We report Nectonema sp. as the first record of a marine nematomorph parasitic in isopod crustaceans. This is also the third record of marine nematomorphs from the North Pacific. Six infected isopods (Natatolana japonensis) collected from 1425 m of depth in the Sea of Japan each contained one to seven (mean 2.33) nematomorphs in the body cavity in the pereon. There was no correlation between the host body length and number of parasites. For Nectonema sp., we describe and illustrate morphological features of the parasitic juvenile stage and present nucleotide sequences for the cytochrome c oxidase subunit I gene (COI or cox1; 451 nt), 18S rRNA gene (1777 nt), and region spanning the internal transcribed spacer 1 (ITS1) and the 28S rRNA gene including the 5.8S rRNA gene and ITS2 (1218 nt in total). In an 18S maximum-likelihood tree that included 24 nematomorph species, Nectonema sp. grouped with N. agile from the northwestern Atlantic; the 18S gene from these two taxa was divergent by 11.8% K2P distance, suggesting that they are different species. Nectonema species may have a broader range of host groups than previously suspected, but may have been previously misidentified as nematode parasites.

Single-strand conformation polymorphism-based genetic characterization of the Cyclospora cayetanensis strains collected from different provinces in Turkey.

INTRODUCTION AND OBJECTIVE: Cyclospora cayetanensis, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of C. cayetanensis and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of C. cayetanensis isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP). MATERIAL AND METHODS: A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining. RESULTS: SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected. CONCLUSIONS: Further comprehensive studies involving more C. cayetanensis-positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

Distribution and diversity of eukaryotic microalgae in Kuwait waters assessed using 18S rRNA gene sequencing.

The microbial communities play a crucial role in ecosystem functioning through interactions among individuals and taxonomic groups in a highly dynamic marine ecosystem. The structure and functioning of the microbial communities are often influenced by the changes in the surrounding environment. Monitoring the microbial diversity of the marine ecosystem helps to understand spatial patterns of microbial community and changes due to season, climate, and various drivers of biological diversity. Kuwait is characterized by an arid environment with a high degree of temperature variation during summer and winter. Our understanding of spatial distribution patterns of microbial communities, their diversity, and the influence of human activities on the degree of changes in the diversity of the microbial community in Kuwait territorial waters remain unclear. In this study, we employed 18S rRNA sequencing to explore marine microalgal community composition and dynamics in seawater samples collected from Kuwait waters over two seasonal cycles across six locations. A total of 448,184 sequences across 36 replicates corresponding to 12 samples from six stations were obtained. The quality-filtered sequences were clustered into 1,293 representative sequences, which were then classified into different eukaryotic taxa. This study reveals that the phytoplankton community in Kuwait waters is diverse and shows significant variations among different taxa during summer and winter. Dinoflagellates and diatoms were the most abundant season-dependent microalgae taxa in Kuwait waters. Alexandrium and Pyrophacus were abundant in summer, whereas Gonyaulax was abundant during the winter. The abundance of Coscinodiscus and Navicula, of the diatom genera, were also dependent upon both seasonal and possible anthropogenic factors. Our results demonstrate the effectiveness of a sequencing-based approach, which could be used to improve the accuracy of quantitative eukaryotic microbial community profiles.

An Introduction to Next Generation Sequencing Bioinformatic Analysis in Gut Microbiome Studies.

The gut microbiome is a microbial ecosystem which expresses 100 times more genes than the human host and plays an essential role in human health and disease pathogenesis. Since most intestinal microbial species are difficult to culture, next generation sequencing technologies have been widely applied to study the gut microbiome, including 16S rRNA, 18S rRNA, internal transcribed spacer (ITS) sequencing, shotgun metagenomic sequencing, metatranscriptomic sequencing and viromic sequencing. Various software tools were developed to analyze different sequencing data. In this review, we summarize commonly used computational tools for gut microbiome data analysis, which extended our understanding of the gut microbiome in health and diseases.

Methods for Identifying Microbial Natural Product Compounds that Target Kinetoplastid RNA Structural Motifs by Homology and De Novo Modeled 18S rRNA.

The development of novel anti-infectives against Kinetoplastids pathogens targeting proteins is a big problem occasioned by the antigenic variation in these parasites. This is also a global concern due to the zoonosis of these parasites, as they infect both humans and animals. Therefore, we need not only to create novel antibiotics, but also to speed up the development pipeline for these antibiotics. This may be achieved by using novel drug targets for Kinetoplastids drug discovery. In this study, we focused our attention on motifs of rRNA molecules that have been created using homology modeling. The RNA is the most ambiguous biopolymer in the kinetoplatid, which carries many different functions. For instance, tRNAs, rRNAs, and mRNAs are essential for gene expression both in the pro-and eukaryotes. However, all these types of RNAs have sequences with unique 3D structures that are specific for kinetoplastids only and can be used to shut down essential biochemical processes in kinetoplastids only. All these features make RNA very potent targets for antibacterial drug development. Here, we combine in silico methods combined with both computational biology and structure prediction tools to address our hypothesis. In this study, we outline a systematic approach for identifying kinetoplastid rRNA-ligand interactions and, more specifically, techniques that can be used to identify small molecules that target particular RNA. The high-resolution optimized model structures of these kineoplastids were generated using RNA 123, where all the stereochemical conflicts were solved and energies minimized to attain the best biological qualities. The high-resolution optimized model's structures of these kinetoplastids were generated using RNA 123 where all the stereochemical conflicts were solved and energies minimized to attain the best biological qualities. These models were further analyzed to give their docking assessment reliability. Docking strategies, virtual screening, and fishing approaches successfully recognized novel and myriad macromolecular targets for the myxobacterial natural products with high binding affinities to exploit the unmet therapeutic needs. We demonstrate a sensible exploitation of virtual screening strategies to 18S rRNA using natural products interfaced with classical maximization of their efficacy in phamacognosy strategies that are well established. Integration of these virtual screening strategies in natural products chemistry and biochemistry research will spur the development of potential interventions to these tropical neglected diseases.

Molecular characterization and phylogenetic analyses of Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970 based on 18S rRNA gene sequence data.

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Srámek-Husek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful.

Urocleidus sayani n. sp. (Monogenea: Dactylogyridae) from Gills of Pirate Perch (Aphredoderus sayanus).

Urocleidus sayani n. sp. is described from the gills of pirate perch (Aphredoderus sayanus) in the Wisconsin backwaters of the upper Mississippi River and was found in samples from the Southeastern United States. Urocleidus sayani n. sp. is the second monogenean described from the pirate perch and the first for this host within Dactylogyridae. The description includes a partial 18S rRNA gene sequence (623 bp), filling a void in sequence data from North American monogeneans.

Identification, genetic variation, and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar.

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo.

Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

Structure of the Maturing 90S Pre-ribosome in Association with the RNA Exosome.

Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.

Phylogenomics from transcriptomic "bycatch" clarify the origins and diversity of avian trypanosomes in North America.

The eukaryotic blood parasite genus Trypanosoma includes several important pathogens of humans and livestock, but has been understudied in wildlife broadly. The trypanosomes that infect birds are in particular need of increased attention, as these parasites are abundant and globally distributed, yet few studies have addressed their evolutionary origins and diversity using modern molecular and analytical approaches. Of specific interest are the deep evolutionary relationships of the avian trypanosomes relative to the trypanosome species that are pathogenic in humans, as well as their species level diversity in regions where they have been understudied such as North America. Here, we address these unresolved areas of study using phylogenomic data for two species of avian trypanosomes that were isolated as "bycatch" from host transcriptome assemblies, as well as a large 18S DNA barcode sequence dataset that includes 143 novel avian Trypanosoma 18S sequences from North America. Using a phylogenomic approach, we find that the avian trypanosomes are nested within a clade of primarily mammalian trypanosomes that includes the human pathogen Trypanosoma cruzi, and are paraphyletic with respect to the ruminant trypanosome Trypanosoma theileri. DNA barcode sequences showed that T. avium and an unidentified small, non-striated trypanosome that was morphologically similar to T. everetti are each represented by highly abundant and divergent 18S haplotypes in North America. Community-level sampling revealed that additional species-level Trypanosoma lineages exist in this region. We compared the newly sequenced DNA barcodes from North America to a global database, and found that avian Trypanosoma 18S haplotypes generally exhibited a marked lack of host specificity with at least one T. avium haplotype having an intercontinental distribution. This highly abundant T. avium haplotype appears to have a remarkably high dispersal ability and cosmopolitan capacity to evade avian host immune defenses, which warrant further study.